Methdos for detecting estradiol by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of estradiol in a sample using mass spectrometry. The methods generally involve ionizing estradiol in a sample and detecting and quantifying the amount of the ion to determine the amount of estradiol in the sample.

FIELD OF THE INVENTION

The invention relates to the detection of estradiol. In a particularaspect, the invention relates to methods for detecting estradiol by massspectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Estradiol [17β-estradiol or Estra-1,3,5(10)-triene-3,17-diol,(17-beta)]or E2 is a C₁₈ steroid hormone with a molecular weight of 272.38daltons. It is a sex hormone labeled as the “female” hormone (alsopresent in males), which is the most potent estrogen of a group ofendogenous steroids, which include estrone (E1) and estriol (E3). Inwomen, mainly the ovaries produce estradiol with secondary production bythe adrenal glands and conversion of steroid precursors into estrogensin fat tissue. Estradiol is responsible for growth of the breast andreproductive epithelia and development of secondary sexualcharacteristics. Normal levels of estradiol provide for properovulation, conception and pregnancy, in addition to promoting healthybone structure and regulating cholesterol levels in females. In men, thetestes and the adrenal glands are the principal source of estradiol.Estradiol is needed for hormonal balance and the function of otherglands.

Methods for detecting specific estradiol ions using mass spectrometryhave been described. For example Nelson R, et al., Clinical Chem 2004,50(2):373-84; Mellon-Nussbaum S, et al., J Biol Chem 1982,257(10):5678-5684; and Xu X, et al., Nature Protocols 2007,2(6):1350-1355 disclose methods for detecting various estradiol ionsusing liquid chromatography and mass spectrometry. These methodsderivatize estradiol prior to detection by mass spectrometry. Methods todetect underivatized estradiol by liquid chromatography/massspectrometry are disclosed in Guo T, et al., Arch Pathol Lab Med 2004,128:469-475; Sun Y, et al., J Am Soc Mass Spectrom 2005, 16(2):271-279;and Diaz-Cruz S, et al., J Mass Spectrom 2003, 38:917-923. Methods todetect estradiol by gas chromatography/mass spectrometry are disclosedin Nachtigall L, et al., Menopause: J of N. Amer. Menopause Society2000, 7(4):243-250; Santen R, et al., Steroids 2007, 72:666-671; DorganJ, et al., Steroids 2002, 67:151-158; Biancotto G, et al., J MassSpectrom 2002, 37(12): 1266-1271; Fedeniuk R W, et al., J Chromatogr BAnalyt Technol Biomed Life Sci 2004, 802(2):307-315; and Biddle S, etal., Anal Chim Acta, 2007, 586(1-2); 115-121.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount ofestradiol in a sample by mass spectrometry, including tandem massspectrometry.

In one aspect, methods are provided for determining the amount ofestradiol in a test sample. The methods may include: (a) purifyingestradiol in the test sample by liquid chromatography; (b) ionizingestradiol in the test sample; and (c) detecting the amount of theestradiol ion(s) by mass spectrometry and relating the amount of thedetected estradiol ion(s) to the amount of estradiol in the test sample.In certain preferred embodiments of this aspect, the limit ofquantitation of the methods is less than or equal to 80 pg/mL; andpreferably, estradiol is not derivatized prior to mass spectrometry. Insome preferred embodiments, the methods include generating one or moreprecursor ions of estradiol in which at least one of the precursor ionshas a mass/charge ratio of 271.14±0.5 or 255.07±0.5. In relatedpreferred embodiments, the methods may include generating one or morefragment ions of an estradiol precursor ion in which at least one of thefragment ions has a mass/charge ratio of 183.10±0.5, 159.20±0.5,145.10±0.5, or 133.20±0.5; preferably one or more fragment ions areselected from the group consisting of ions with a mass/charge ratio of183.10±0.5 and 133.20±0.5. In certain preferred embodiments of theaspect, the test sample is body fluid. In some preferred embodiments,the methods may include adding an agent to the test sample in an amountsufficient to free estradiol from a protein that may be present in thetest sample. In related preferred embodiments, the methods may includeacidifying the test sample; preferably acidifying before ionizing; morepreferably acidifying before purifying; preferably acidifying withformic acid. In different preferred embodiments, the methods may includeadding a salt to the test sample in an amount sufficient to freeestradiol from a protein that may be present in the test sample;preferably adding before ionizing; more preferably adding beforepurifying; preferably adding ammonium sulfate. In particularly preferredembodiments, 100 mM ammonium sulfate is added to the test sample for afinal concentration of ˜57 mM ammonium sulfate in the test sample.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Samples are purified herein by various means to allow removalof one or more interfering substances, e.g., one or more substances thatwould interfere with the detection of selected estradiol parent anddaughter ions by mass spectrometry.

As used herein, the term “test sample” refers to any sample that maycontain estradiol. As used herein, the term “body fluid” means any fluidthat can be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “derivatizing” means reacting two molecules toform a new molecule. Derivatizing agents may include isothiocyanategroups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm. As used in this context, the term“about” means ±10%. In a preferred embodiment the column containsparticles of about 60 μm in diameter.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means ±10%. In apreferred embodiment the analytical column contains particles of about 4μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); andMerchant and Weinberger, Electrophoresis 21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “E1” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectroscopy methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “Atmospheric Pressure Photoionization” or “APPI” as used hereinrefers to the form of mass spectroscopy where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a precision of 20% and anaccuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is defined arbitrarily as 2 standard deviations (SD) fromthe zero concentration.

As used herein, an “amount” of estradiol in a body fluid sample refersgenerally to an absolute value reflecting the mass ofestradioldetectable in volume of body fluid. However, an amount also contemplatesa relative amount in comparison to another estradiol amount. Forexample, an amount ofestradiol in a body fluid can be an amount which isgreater than a control or normal level of estradiol normally present.

In a second aspect, methods are provided for determining the amount ofestradiol in a body fluid sample by tandem mass spectrometry thatinclude: (a) purifying estradiol in the body fluid sample by liquidchromatography; (b) generating a precursor ion ofestradiol having amass/charge ratio of 255.07±0.5; (c) generating one or more fragmentions of the precursor ion in which at least one of the fragment ions hasa mass/charge ratio of 133.20±0.5; and (d) detecting the amount of oneor more of the ions generated in step (b) or (c) or both and relatingthe detected ions to the amount of estradiol in the body fluid sample.In some preferred embodiments, the limit of quantitation of the methodsis less than or equal to 80 pg/mL. In other preferred embodiments,estradiol is not derivatized prior to mass spectrometry. In certainpreferred embodiments, the methods may further include generating one ormore fragment ions of an estradiol precursor ion in which at least oneof the fragment ions has a mass/charge ratio of 159.20±0.5. In somepreferred embodiments, the methods may include adding an agent to thebody fluid sample in an amount sufficient to free estradiol from aprotein that may be present in the body fluid sample. In relatedpreferred embodiments, the methods may include acidifying the body fluidsample; preferably acidifying before ionizing; more preferablyacidifying before purifying; preferably acidifying with formic acid.

In a third aspect, methods are provided for determining the amount ofestradiol in a body fluid sample by tandem mass spectrometry thatinclude: (a) purifying estradiol in the body fluid sample by liquidchromatography; (b) generating a precursor ion of estradiol having amass/charge ratio of 271.14±0.5; (c) generating one or more fragmentions of the precursor ion in which at least one of the fragment ions hasa mass/charge ratio of 183.10±0.5; and (d) detecting the amount of oneor more of the ions generated in step (b) or (c) or both and relatingthe detected ions to the amount of estradiol in the body fluid sample.In some preferred embodiments, the limit of quantitation of the methodsis less than or equal to 80 pg/mL. In other preferred embodiments,estradiol is not derivatized prior to mass spectrometry. In certainpreferred embodiments, the methods may further include generating one ormore fragment ions of an estradiol precursor ion in which at least oneof the fragment ions has a mass/charge ratio of 145.10±0.5. In somepreferred embodiments, the methods may include adding an agent to thebody fluid sample in an amount sufficient to free estradiol from aprotein that may be present in the body fluid sample. In relatedpreferred embodiments, the methods may include adding a salt agent tothe body fluid sample; preferably adding before ionizing; morepreferably adding before purifying; preferably adding ammonium sulfate.In particularly preferred embodiments, 100 mM ammonium sulfate is addedto the body fluid sample for a final concentration of ˜57 mM ammoniumsulfate in the body fluid sample.

In a fourth aspect, methods are provided for determining the amount ofestradiol in a test sample that include: (a) acidifying the test samplewith an agent in an amount sufficient to free estradiol from a proteinthat may be present in the test sample; (b) purifying estradiol in thetest sample by liquid chromatography; (c) ionizing estradiol in the testsample to produce one or more ions detectable by tandem massspectrometry; and (d) detecting the amount of the estradiol ion(s) bytandem mass spectrometry in positive ion mode and relating the amount ofthe detected estradiol ion(s) to the amount of estradiol in the testsample. In certain preferred embodiments, the test sample is body fluid.In some preferred embodiments, the limit of quantitation of the methodsis less than or equal to 80 pg/mL. In other preferred embodiments,estradiol is not derivatized prior to mass spectrometry. In somepreferred embodiments, the methods include generating one or moreprecursor ions of estradiol in which at least one of the precursor ionshas a mass/charge ratio of 255.07±0.5. In related preferred embodiments,the methods may include generating one or more fragment ions of anestradiol precursor ion in which at least one of the fragment ions has amass/charge ratio of 159.20±0.5 or 133.20±0.5. In some preferredembodiments, the methods may include acidifying the test sample beforeionizing; more preferably acidifying before purifying; preferablyacidifying with formic acid.

In a fifth aspect, methods are provided for determining the amount ofestradiol in a test sample that include: (a) adding an agent to the testsample in an amount sufficient to free estradiol from a protein that maybe present in the test sample; (b) purifying estradiol in the testsample by liquid chromatography; (c) ionizing the purified estradiol inthe test sample to produce one or more ions detectable by tandem massspectrometry; and (d) detecting the amount of the estradiol ion(s) bytandem mass spectrometry in negative ion mode and relating the amount ofthe detected estradiol ion(s) to the amount of estradiol in the testsample. In certain preferred embodiments, the test sample is body fluid.In some preferred embodiments, the limit of quantitation of the methodsis less than or equal to 80 pg/mL. In other preferred embodiments,estradiol is not derivatized prior to mass spectrometry. In somepreferred embodiments, the methods include generating one or moreprecursor ions of estradiol in which at least one of the precursor ionshas a mass/charge ratio of 271.14±0.5. In related preferred embodiments,the methods may include generating one or more fragment ions of anestradiol precursor ion in which at least one of the fragment ions has amass/charge ratio of 183.10±0.5 or 145.10±0.5. In other preferredembodiments, the methods may include adding a salt agent to the testsample, preferably adding before ionizing; more preferably adding beforepurifying, preferably adding ammonium sulfate. In particularly preferredembodiments, 100 mM ammonium sulfate is added to the test sample for afinal concentration of ˜57 mM ammonium sulfate in the test sample.

In some preferred embodiments, estradiol may be derivatized prior tomass spectrometry, however, in certain preferred embodiments; samplepreparation excludes the use of derivatization.

In certain preferred embodiments of the above aspects, liquidchromatography is performed using HTLC and HPLC, preferably HTLC is usedin conjunction with HPLC, however other methods can be used that includefor example, protein precipitation and purification in conjunction withHPLC.

Preferred embodiments utilize high performance liquid chromatography(HPLC), alone or in combination with one or more purification methods,for example HTLC or protein precipitation, to purify estradiol insamples.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in negative ion mode. Alternatively, massspectrometry is performed in positive ion mode. In particularlypreferred embodiments, estradiol is measured using both positive andnegative ion mode. In certain preferred embodiments, estradiol ismeasured using APCI or ESI in either positive or negative mode.

In preferred embodiments of the above aspects, both glucuronidated andnon-glucuronidated estradiol present in the body fluid sample aredetected and measured.

In preferred embodiments, the estradiol ions detectable in a massspectrometer are selected from the group consisting of ions with amass/charge ratio (m/z) of 271.14±0.5, 255.07±0.5, 183.10±0.5,159.20±0.5, 145.10±0.5, and 133.20±0.5; the latter four being fragmentions of the precursor ions. In particularly preferred embodiments, theprecursor ions have a mass/charge ratio of 271.14±0.5 or 255.07±0.5, andthe fragment ions have a mass/charge ratio of 183.10±0.5 or 133.20±0.5.

In preferred embodiments, a separately detectable internal estradiolstandard is provided in the sample, the amount of which is alsodetermined in the sample. In these embodiments, all or a portion of boththe endogenous estradiol and the internal standard present in the sampleis ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each are detected bymass spectrometry.

A preferred internal estradiol standard is 2,4,16,16,17-d₅ estradiol. Inpreferred embodiments, the internal estradiol standard ions detectablein a mass spectrometer are selected from the group consisting of ionswith a mass/charge ratio of 276.15±0.5, 260.10±0.5, 187.10±0.5,161.10±0.5, 147.10±0.5, and 135.10±0.5. In particularly preferredembodiments, a precursor ion of the internal estradiol standard isselected from the group consisting of ions having a mass/charge ratio of276.15±0.5 and 260.10±0.5; and one or more fragment ions is selectedfrom the group consisting of ions having a mass/charge ratio of187.10±0.5, 161.10±0.5, 147.10±0.5, and 135.10±0.5.

In preferred embodiments, the presence or amount of the estradiol ion isrelated to the presence or amount of estradiol in the test sample bycomparison to a reference such as 2,4,16,16,17-d₅ estradiol.

In one embodiment, the methods involve the combination of liquidchromatography with mass spectrometry. In a preferred embodiment, theliquid chromatography is HPLC. A preferred embodiment utilizes HPLCalone or in combination with one or more purification methods such asfor example HTLC or protein purification, to purify estradiol insamples. In another preferred embodiment, the mass spectrometry istandem mass spectrometry (MS/MS).

In certain preferred embodiments of the aspects disclosed herein, thelimit of quantitation (LOQ) of estradiol is less than or equal to 80pg/mL; preferably less than or equal to 75 pg/mL; preferably less thanor equal to 50 pg/mL; preferably less than or equal to 25 pg/mL;preferably less than or equal to 10 pg/mL; preferably less than or equalto 5 pg/mL; preferably less than or equal to 4.5 pg/mL; preferably lessthan or equal to 4 pg/mL; preferably less than or equal to 3.5 pg/mL;preferably less than or equal to 3 pg/mL; preferably less than or equalto 2.5 pg/mL; preferably 2 pg/mL.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.5 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the linearity of the quantitation of estradiol in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 6.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for detecting and quantifying estradiol in a testsample. The methods utilize liquid chromatography (LC), most preferablyHTLC in conjunction with HPLC, to perform an initial purification ofselected analytes, and combine this purification with unique methods ofmass spectrometry (MS), thereby providing a high-throughput assay systemfor detecting and quantifying estradiol in a test sample. The preferredembodiments are particularly well suited for application in largeclinical laboratories. Estradiol methods are provided that have enhancedspecificity and are accomplished in less time and with less samplepreparation than required in other estradiol assays.

In preferred embodiments, the limit of detection (LOD) of estradiol intest samples is less than or equal to 80 pg/mL; preferably less than orequal to 75 pg/mL; preferably less than or equal to 50 pg/mL; preferablyless than or equal to 25 pg/mL; preferably less than or equal to 10pg/mL; preferably less than or equal to 5 pg/mL; preferably less than orequal to 4.5 pg/mL; preferably less than or equal to 4 pg/mL; preferablyless than or equal to 3.5 pg/mL; preferably less than or equal to 3pg/mL; preferably less than or equal to 2.5 pg/mL; preferably 2 pg/mL.

Suitable test samples include any test sample that may contain theanalyte of interest. For example, samples obtained during themanufacture of synthetic estradiol may be analyzed to determine thecomposition and yield of the manufacturing process. In some preferredembodiments, a sample is a biological sample; that is, a sample obtainedfrom any biological source, such as an animal, a cell culture, an organculture, etc. In certain preferred embodiments samples are obtained froma mammalian animal, such as a dog, cat, horse, etc. Particularlypreferred mammalian animals are primates, most preferably male or femalehumans. Particularly preferred samples include blood, plasma, serum,hair, muscle, urine, saliva, tear, cerebrospinal fluid, or other tissuesample. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition. The test sample is preferably obtained from a patient, forexample, blood serum.

Sample Preparation for Mass Spectrometry

In women, approximately 1.3% of estradiol circulates in free form withthe remainder bound to sex hormone binding globulin (SHBG) and toalbumin. Estradiol binds to SHBG with high affinity (approximately 40%)or to albumin with lower affinity.

Methods that may be used to enrich in estradiol relative to othercomponents in the sample (e.g. protein) include for example, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateextraction and methanol extraction, and the use of chaotropic agents orany combination of the above or the like.

Various methods may be used to disrupt the interaction between estradioland protein prior to chromatography and or MS sample analysis so thatthe analysis can be directed to the total amount of estradiol in thesample (e.g., free estradiol and estradiol bound to protein). Proteinprecipitation is one preferred method of preparing a test sample,especially a biological test sample, such as serum or plasma. Suchprotein purification methods are well known in the art, for example,Poison et al., Journal of Chromatography B 785:263-275 (2003), describesprotein precipitation techniques suitable for use in the methods.Protein precipitation may be used to remove most of the protein from thesample leaving estradiol in the supernatant. The samples may becentrifuged to separate the liquid supernatant from the precipitatedproteins. The resultant supernatant may then be applied to liquidchromatography and subsequent mass spectrometry analysis. In certainembodiments, the use of protein precipitation such as for example,acetonitrile protein precipitation, obviates the need for highturbulence liquid chromatography (HTLC) or other on-line extractionprior to HPLC and mass spectrometry. Accordingly in such embodiments,the method involves (1) performing a protein precipitation of the sampleof interest; and (2) loading the supernatant directly onto the HPLC-massspectrometer without using on-line extraction or high turbulence liquidchromatography (HTLC).

In other preferred embodiments, estradiol may be released from a proteinwithout having to precipitate the protein. For example, formic acid or40% ethanol in water may be added to the sample to disrupt interactionbetween a protein and estradiol. Alternatively, ammonium sulfate may beadded to the sample to disrupt ionic interactions between a carrierprotein and estradiol without precipitating the carrier protein.

In some preferred embodiments, HTLC, alone or in combination with one ormore purification methods, may be used to purify estradiol prior to massspectrometry. In such embodiments samples may be extracted using an HTLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLCcolumn prior to ionization. Because the steps involved in thesechromatography procedures can be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized. This feature can result in savings of time andcosts, and eliminate the opportunity for operator error.

It is believed that turbulent flow, such as that provided by HTLCcolumns and methods, may enhance the rate of mass transfer, improvingseparation characteristics. HTLC columns separate components by means ofhigh chromatographic flow rates through a packed column containing rigidparticles. By employing high flow rates (e.g., 3-5 mL/min), turbulentflow occurs in the column that causes nearly complete interactionbetween the stationary phase and the analyte(s) of interest. Anadvantage of using HTLC columns is that the macromolecular build-upassociated with biological fluid matrices is avoided since the highmolecular weight species are not retained under the turbulent flowconditions. HTLC methods that combine multiple separations in oneprocedure lessen the need for lengthy sample preparation and operate ata significantly greater speed. Such methods also achieve a separationperformance superior to laminar flow (HPLC) chromatography. HTLC allowsfor direct injection of biological samples (plasma, urine, etc.). Directinjection is difficult to achieve in traditional forms of chromatographybecause denatured proteins and other biological debris quickly block theseparation columns. HTLC also allows for very low sample volume of lessthan 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL,preferably 0.1 mL.

Examples of HTLC applied to sample preparation prior to analysis by massspectrometry have been described elsewhere. See, e.g., Zimmer et al., J.Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of themethod, samples are subjected to protein precipitation as describedabove prior to loading on the HTLC column; in alternative preferredembodiments, the samples may be loaded directly onto the HTLC withoutbeing subjected to protein precipitation. Preferably, HTLC is used inconjunction with HPLC to extract and purify estradiol without the samplebeing subjected to protein precipitation. In related preferredembodiments, the purifying step involves (i) applying the sample to anHTLC extraction column, (ii) washing the HTLC extraction column underconditions whereby estradiol is retained by the column, (iii) elutingretained estradiol from the HTLC extraction column, (iv) applying theretained material to an analytical column, and (v) eluting purifiedestradiol from the analytical column. The HTLC extraction column ispreferably a large particle column. In various embodiments, one of moresteps of the methods may be performed in an on-line, automated fashion.For example, in one embodiment, steps (i)-(v) are performed in anon-line, automated fashion. In another, the steps of ionization anddetection are performed on-line following steps (i)-(v).

Liquid chromatography (LC) including high-performance liquidchromatography (HPLC) relies on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packings in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess. HPLC has been successfully applied to the separation ofcompounds in biological samples but a significant amount of samplepreparation is required prior to the separation and subsequent analysiswith a mass spectrometer (MS), making this technique labor intensive. Inaddition, most HPLC systems do not utilize the mass spectrometer to itsfullest potential, allowing only one HPLC system to be connected to asingle MS instrument, resulting in lengthy time requirements forperforming a large number of assays.

Various methods have been described for using HPLC for sample clean-upprior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000); and Salm et al., Clin.Therapeutics 22 Supl. B:B71-B85 (2000).

One of skill in the art may select HPLC instruments and columns that aresuitable for use with estradiol. The chromatographic column typicallyincludes a medium (i.e., a packing material) to facilitate separation ofchemical moieties (i.e., fractionation). The medium may include minuteparticles. The particles include a bonded surface that interacts withthe various chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded surface. Alkyl bonded surfaces may include C-4,C-8, C-12, or C-18 bonded alkyl groups, preferably C-18 bonded groups.The chromatographic column includes an inlet port for receiving a sampleand an outlet port for discharging an effluent that includes thefractionated sample. In one embodiment, the sample (or pre-purifiedsample) is applied to the column at the inlet port, eluted with asolvent or solvent mixture, and discharged at the outlet port. Differentsolvent modes may be selected for eluting the analyte(s) of interest.For example, liquid chromatography may be performed using a gradientmode, an isocratic mode, or a polytyptic (i.e. mixed) mode. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluent (also known as a “mobile phase”), elution mode,gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, the HTLC may be followed by HPLC on ahydrophobic column chromatographic system. In certain preferredembodiments, a TurboFlow Cyclone P® polymer-based column from CohesiveTechnologies (60 μm particle size, 50×1.0 mm column dimensions, 100 Åpore size) is used. In related preferred embodiments, a SynergiPolar-RP® ether-linked phenyl, analytical column from Phenomenex Inc (4μm particle size, 150×2.0 mm column dimensions, 80 Å pore size) withhydrophilic endcapping is used. In certain preferred embodiments, HTLCand HPLC are performed using HPLC Grade Ultra Pure Water and 100%methanol as the mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In certain preferred embodiments, estradiol present in a test sample maybe purified prior to ionization. In particularly preferred embodimentsthe chromatography is not gas chromatography. Preferably, the methodsare performed without subjecting estradiol, to gas chromatography priorto mass spectrometric analysis.

Detection and Quantitation by Mass Spectrometry

In various embodiments, estradiol present in a test sample may beionized by any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

In preferred embodiments, estradiol is ionized by electrosprayionization (ESI) in positive or negative mode. In related preferredembodiments, estradiol ion is in a gaseous state and the inert collisiongas is argon or nitrogen. In alternative preferred embodiments,estradiol is ionized by atmospheric pressure chemical ionization (APCI)in positive or negative mode.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio. Suitable analyzers for determining mass-to-chargeratios include quadrupole analyzers, ion traps analyzers, andtime-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected i.e., usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of estradiol. Methods of generating and using such standardcurves are well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotope ofestradiol may be used as an internal standard; in certain preferredembodiments the standard is d₅-estradiol. Numerous other methods forrelating the amount of an ion to the amount of the original moleculewill be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition”. Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, estradiol is detected and/orquantified using MS/MS as follows. The samples are subjected to liquidchromatography, preferably HTLC followed by HPLC, the flow of liquidsolvent from the chromatographic column enters the heated nebulizerinterface of an MS/MS analyzer and the solvent/analyte mixture isconverted to vapor in the heated tubing of the interface. The analyte(e.g., estradiol), contained in the nebulized solvent, is ionized by thecorona discharge needle of the interface, which applies a large voltageto the nebulized solvent/analyte mixture. The ions, e.g. precursor ions,pass through the orifice of the instrument and enter the firstquadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowingselection of ions (i.e., “precursor” and “fragment” ions) based on theirmass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cell,where ions are fragmented. The first quadrupole of the mass spectrometer(Q1) selects for molecules with the mass to charge ratios of estradiol.Precursor ions with the correct mass/charge ratios of estradiol areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. This process is calledcollision activated dissociation (CAD). The fragment ions generated arepassed into quadrupole 3 (Q3), where the fragment ions of estradiol areselected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode. Using standard methods well known in the art, one of ordinaryskill is capable of identifying one or more fragment ions of aparticular precursor ion of estradiol that may be used for selection inquadrupole 3 (Q3).

If the precursor ion of estradiol includes an alcohol or amine group,fragment ions are commonly formed that represent dehydration ordeamination of the precursor ion, respectfully. In the case of precursorions that include an alcohol group, such fragment ions formed bydehydration are caused by a loss of one or more water molecules from theprecursor ion (i.e., where the difference in mass to charge ratiobetween the precursor ion and fragment ion is about 18 for the loss ofone water molecule, or about 36 for the loss of two water molecules,etc.). In the case of precursor ions that include an amine group, suchfragment ions formed by deamination are caused by a loss of one or moreammonia molecules (i.e. where the difference in mass to charge ratiobetween the precursor ion and fragment ion is about 17 for the loss ofone ammonia molecule, or about 34 for the loss of two ammonia molecules,etc.). Likewise, precursor ions that include one or more alcohol andamine groups commonly form fragment ions that represent the loss of oneor more water molecules and/or one or more ammonia molecules (i.e.,where the difference in mass to charge ratio between the precursor ionand fragment ion is about 35 for the loss of one water molecule and theloss of one ammonia molecule). Generally, the fragment ions thatrepresent dehydrations or deaminations of the precursor ion are notspecific fragment ions for a particular analyte. Accordingly, inpreferred embodiments of the invention, MS/MS is performed such that atleast one fragment ion of estradiol is detected that does not representonly a loss of one or more water molecules and/or a loss of one or moreammonia molecules from the precursor ion.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte (estradiol)of interest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of estradiol. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., estradiol, using calibrationstandard curves based on peaks of one or more ions of an internalmolecular standard, such as d₅-estradiol.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample and Reagent Preparation

Blood was collected in a Vacutainer with no additives and allowed toclot 30 minutes at room temperature, 18° to 25° C. Samples thatexhibited gross hemolysis and/or lipemia were excluded.

An estradiol stock standard of 1 mg/mL in methanol was prepared andfurther diluted in methanol to prepare an estradiol intermediate stockstandard of 1,000,000 pg/mL, which was used to prepare two estradiolworking standards of 10,000 pg/mL, diluted in either methanol forstandard A or in stripped serum for standard B.

Deuterated methanol (methyl-d₁ alcohol; Fisher Cat. No. AC29913-1000 orequivalent) was used to prepare a 1 mg/mL d₅-estradiol stock standard(2,4,16,16,17-d₅ estradiol), which was used to prepare a 1,000,000 pg/mLintermediate stock standard in deuterated methanol. The d₅-estradiolintermediate stock standard was used to prepare a working d₅-estradiolinternal standard of 5000 pg/mL in DI water: 1 mL of the d₅-estradiolintermediate stock standard was diluted to volume with DI water in a 200mL volumetric flask.

A 20% formic acid solution was prepared by adding 50 mL of formic acid(˜98% pure Aldrich Cat. No. 06440 or equivalent) to a 250 mL volumetricflask, which was diluted to volume with ultrapure HPLC-grade water. A100 mM ammonium sulfate solution in DI water was prepared by adding 13.2gram of ammonium sulfate powder (CAS#7783-20-2 Fisher Cat. No: A702-500)to a 1000 mL volumetric flask, which was diluted to volume with DIwater.

All calibrators/standards used in each run were prepared fresh weeklyfrom series of dilutions of frozen aliquots of 10,000 pg/mL estradiolstandard in stripped serum. The standards were prepared from highestconcentration to the lowest with a final total volume for each standardof 10 mL.

Example 2 Extraction of Estradiol from Serum Using Liquid Chromatography

Liquid chromatography (LC) samples were prepared by pipetting 200 μL ofstandards, controls, or patient samples into a 96-well plate. If run onpositive ion mode, 300 μL of 20% formic acid were delivered to each wellfor a final concentration of ˜11% (V/V). If run on negative ion mode,300 μL of 100 mM ammonium sulfate solution were added to each well for afinal concentration of ˜57 mM ammonium sulfate. In addition, 25 μL ofthe 5000 pg/mL d₅-estradiol standard were added to each well. Thesamples were incubated at room temperature for 30 to 45 minutes prior toLC.

Liquid chromatography was performed with a Cohesive Technologies AriaTX-4 HTLC system using Aria OS V 1.5 or newer software. An autosamplerwash solution was prepared using 30% acetonitrile, 30% methanol, 30%isopropanol, and 10% acetone (v/v).

The HTLC system automatically injected 75 μL of the above preparedsamples into a TurboFlow column (50×1.0 mm, 60 m Cyclone P column fromCohesive Technologies) packed with large particles. The samples wereloaded at a high flow rate (5 mL/min, loading reagent 100% DI) to createturbulence inside the extraction column. This turbulence ensuredoptimized binding of estradiol to the large particles in the column andthe passage of residual protein and debris to waste.

Following loading, the flow direction was reversed and the sample elutedoff to the analytical column (Phenomenex analytical column, SynergiPolar-RP® 150×2.0 mm, 4 m column) with 200 μL of 100% methanol in theloop. A binary HPLC gradient was applied to the analytical column, toseparate estradiol from other analytes contained in the sample. Mobilephase A was Ultra Pure Water (HPLC grade) and mobile phase B was 100%methanol. The HPLC gradient started with a 40% organic gradient whichramped to 100% in approximately 5.33 minutes. The analytes eluted offthe HPLC column at 77% methanol gradient. The separated sample was thensubjected to MS/MS for quantitation of estradiol.

To determine interference with other molecules, blank sera was spikedwith 1000 pg/mL each of steroids of the same weight such as 16-βestradiol and 17-α estradiol as well as the following steroids: estrone,estriol, testosterone, 17-α hydroxyprogesterone, progesterone,androstenedione, 17-β estradiol glucuronide, 17-β estradriol sulfate,and dihydroxytestosterone. The samples were subject to LC. None of thesteroids co-eluted with estradiol except for 17-α estradiol, whichpartially interfered with 17-β estradiol but had sufficiently differentretention time to allow the operator to determine its presence readily.

Example 3 Detection and Quantitation of Estradiol by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs all fromThermoElectron were used in the Examples described herein: Tune Master V1.2 or newer, Xcalibur V 2.0 SRI or newer, TSQ Quantum 1.4 or newer,LCQuan V 2.0 or newer, and XReport 1.0 or newer. Liquid solvent/analyteexiting the analytical HPLC column flowed to the heated nebulizerinterface of a Thermo Finnigan MS/MS analyzer. The solvent/analytemixture was converted to vapor in the heated tubing of the interface.Analytes in the nebulized solvent were ionized by the corona dischargeneedle of the interface, which applied voltage to the nebulizedsolvent/analyte mixture.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of either 271.14±0.5 m/z or 255.07±0.5 m/z. Ionsentering Quadrupole 2 (Q2) collided with argon gas to generate ionfragments, which were passed to quadrupole 3 (Q3) for further selection.Simultaneously, the same process using isotope dilution massspectrometry was carried out with an internal standard, a 5-deuteratedestradiol molecule. The following mass transitions were used fordetection and quantitation during validation on positive polarity.

TABLE 1 Mass Transitions for Estradiol (Positive Polarity) Precursor IonProduct Ion Analyte (m/z) (m/z) Estradiol 255.07 133.20 & 159.202,4,16,16,17-d₅ Estradiol 260.10 135.10 & 161.10

The following mass transitions were used for detection and quantitationduring validation on negative polarity.

TABLE 2 Mass Transitions for Estradiol (Negative Polarity) Precursor IonProduct Ion Analyte (m/z) (m/z) Estradiol 271.14 145.10 & 183.102,4,16,16,17-d₅ Estradiol 276.15 147.10 & 187.10

Example 4 Intra-Assay and Inter-Assay Precision and Accuracy

Three quality control (QC) pools were prepared from charcoal strippedserum, spiked with estradiol to a concentration of 10, 200, and 800pg/mL.

Ten aliquots from each of the three QC pools were analyzed in a singleassay to determine the reproducibility (CV) of a sample within an assay.The following values were determined:

TABLE 3 Intra-Assay Variation and Accuracy Level I Level II Level III(10 pg/mL) (200 pg/mL) (800 pg/mL) Mean 12 203 804 Stdev 0.8 14.8 24.2CV 7.4% 7.3% 3.0% Accuracy  115%   102%   101%

Ten aliquots from each of the three QC pools were assayed over 5 days todetermine the reproducibility (RSD %) between assays. The followingvalues were determined:

TABLE 4 Inter-Assay Variation and Accuracy Level I Level II Level III(10 pg/mL) (200 pg/mL) (800 pg/mL Mean 11 209 797 Stdev 2.0 26.8 81.6RSD (%) 17.6 12.8 10.2 Accuracy (%) 112.9 104.3 99.7

Example 5 Analytical Sensitivity Limit of Detection (LOD) and Limit ofQuantitation (LOQ)

The estradiol zero standard was run in 10 replicates to determine thelimit of detection of the assay, which is the point at which themeasured value is larger than the uncertainty associated with it. TheLOD was defined arbitrarily as 2 standard deviations (SD) from the zeroconcentration. The resulting peak area ratios for the zero standard werestatistically analyzed with a mean value of 0.021 and a SD of 0.006. TheLOD for the estradiol assay was 2.0 pg/mL.

To determine the limit of quantitation with a precision of 20% and anaccuracy of 80% to 120%, five different samples at concentrations closeto the expected LOQ were assayed and the reproducibility determined foreach. The LOQ for the estradiol assay was defined at 2.0 pg/mL.

Example 6 Assay Reportable Range and Linearity

To establish the linearity of estradiol detection in the assay, oneblank assigned as zero standard and 11 spiked serum standards wereprepared and analyzed on 5 separate days. A quadratic regression fromfive consecutive runs yielded coefficient correlations of 0.995 orgreater, with an accuracy of ±20% revealing a quantifiable linear rangeof 2 to 2000 pg/mL.

Example 7 Matrix Specificity

Matrix specificity was evaluated using water, stripped serum, and pooledserum to determine whether patient samples could be diluted in a linearfashion. The samples were run in duplicate following a calibration run.The accuracy was as follows:

TABLE 5 Matrix Specificity Accuracy Stripped Pooled Water Serum Serum1:2 Dilution 120% 94% 147% 1:4 Dilution  38% 85%  94%

Example 8 Recovery

To determine the ability to recover estradiol from spiked samples, twopatient samples of known concentrations were spiked with 3 levels (low,medium, high) of estradiol. The recovery was calculated by dividing thespiked amount by the expected concentration. The mean recoveries were92%, 108% and 98% for low, mid and high levels respectively.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofestradiol in a test sample said method comprising: (a) purifyingestradiol from said test sample by liquid chromatography; (b) ionizingthe purified estradiol from said test sample to produce one or more ionsdetectable by tandem mass spectrometry; and (c) detecting the amount ofthe estradiol ion(s) by tandem mass spectrometry, wherein the amount ofthe estradiol ion(s) is related to the amount of estradiol in said testsample; wherein the limit of quantitation of the method is less than orequal to 80 pg/mL and estradiol is not derivatized prior to massspectrometry.
 2. The method of claim 1, wherein said liquidchromatography is high performance liquid chromatography (HPLC).
 3. Themethod of claim 1, wherein said purifying further comprises highturbulence liquid chromatography (HTLC) followed by high performanceliquid chromatography (HPLC).
 4. The method of claim 1, wherein saidestradiol ions comprise one or more ions selected from the groupconsisting of ions with a mass/charge ratio of 271.14±0.5, 255.07±0.5,183.10±0.5, 159.20±0.5, 145.10±0.5, and 133.20±0.5.
 5. The method ofclaim 1, wherein said ionizing comprises generating a precursor ionselected from the group consisting of ions with a mass/charge ratio of271.14±0.5 and 255.07±0.5, and generating one or more fragment ionsselected from the group consisting of ions with a mass/charge ratio of183.10±0.5, 159.20±0.5, 145.10±0.5, and 133.20±0.5.
 6. The method ofclaim 1, wherein an agent is added to said test sample in an amountsufficient to free estradiol from a protein that may be present in saidtest sample prior to said ionizing step.
 7. The method of claim 6,wherein said agent is added in an amount sufficient to acidify said testsample.
 8. The method of claim 7, wherein said agent is formic acid. 9.The method of claim 6, wherein said agent is a salt.
 10. The method ofclaim 9, wherein said salt is ammonium sulfate.
 11. The method of claim1, wherein both glucuronidated and non-glucuronidated estradiol presentin said test sample are detected and measured by the method.
 12. Themethod of claim 1, wherein said test sample is body fluid.
 13. A methodfor determining the amount of estradiol in a body fluid sample by tandemmass spectrometry said method comprising: (a) purifying estradiol fromsaid body fluid sample by liquid chromatography; (b) generating aprecursor ion of said estradiol having a mass/charge ratio of255.07±0.5; (c) generating one or more fragment ions of said precursorion, wherein at least one of said one or more fragment ions comprise anion fragment having a mass/charge ratio of 133.20±0.5; and (d) detectingthe amount of one or more of said ions generated in step (b) or (c) orboth and relating the detected ions to the amount of said estradiol insaid body fluid sample.
 14. The method of claim 13, wherein said methodhas a limit of quantitation less than or equal to 80 pg/mL.
 15. Themethod of claim 13, wherein said estradiol is not derivatized prior tomass spectrometry.
 16. The method of claim 13, wherein said liquidchromatography is high performance liquid chromatography (HPLC).
 17. Themethod of claim 13, wherein said purifying further comprises highturbulence liquid chromatography (HTLC) followed by high performanceliquid chromatography (HPLC).
 18. The method of claim 13, wherein saidprecursor ion has a mass/charge ratio of 255.07±0.5, and said one ormore fragment ions comprise a fragment ion with a mass/charge ratio of159.20±0.5.
 19. The method of claim 13, wherein an agent is added tosaid body fluid sample in an amount sufficient to free estradiol from aprotein that may be present in said body fluid sample prior to saidionizing step.
 20. The method of claim 19, wherein said agent is addedin an amount sufficient to acidify said body fluid sample.
 21. Themethod of claim 20, wherein said agent is formic acid.
 22. The method ofclaim 13, wherein both glucuronidated and non-glucuronidated estradiolpresent in said body fluid are detected and measured by the method. 23.A method for determining the amount of estradiol in a body fluid sampleby tandem mass spectrometry said method comprising: (a) purifyingestradiol from said body fluid sample by liquid chromatography; (b)generating a precursor ion of said estradiol having a mass/charge ratioof 271.14±0.5; (c) generating one or more fragment ions of saidprecursor ion, wherein at least one of said one or more fragment ionscomprise an ion fragment having a mass/charge ratio of 183.10±0.5; and(d) detecting the amount of one or more of said ions generated in step(b) or (c) or both and relating the detected ions to the amount of saidestradiol in said body fluid sample.
 24. The method of claim 23, whereinsaid method has a limit of quantitation less than or equal to 80 pg/mL.25. The method of claim 23, wherein said estradiol is not derivatizedprior to mass spectrometry.
 26. The method of claim 23, wherein saidliquid chromatography is high performance liquid chromatography (HPLC).27. The method of claim 23, wherein said purifying further compriseshigh turbulence liquid chromatography (HTLC) followed by highperformance liquid chromatography (HPLC).
 28. The method of claim 23,wherein said precursor ion has a mass/charge ratio of 271.14±0.5, andsaid one or more fragment ions comprise a fragment ion with amass/charge ratio of 145.10±0.5.
 29. The method of claim 23, wherein anagent is added to said body fluid sample in an amount sufficient to freeestradiol from a protein that may be present in said test sample priorto said ionizing step.
 30. The method of claim 29, wherein said agent isa salt.
 31. The method of claim 30, wherein said salt is ammoniumsulfate.
 32. The method of claim 23, wherein both glucuronidated andnon-glucuronidated estradiol present in said body fluid sample aredetected and measured by the method.
 33. A method for determining theamount of estradiol in a test sample said method comprising: (a)acidifying said test sample with an agent in an amount sufficient tofree estradiol from a protein that may be present in said test sample;(b) purifying estradiol from said test sample by liquid chromatography;(c) ionizing the purified estradiol from said test sample to produce oneor more ions detectable by tandem mass spectrometry; and (d) detectingthe amount of the estradiol ion(s) by tandem mass spectrometry inpositive ion mode, wherein the amount of the estradiol ion(s) is relatedto the amount of estradiol in said test sample.
 34. The method of claim33, wherein said method has a limit of quantitation less than or equalto 80 pg/mL.
 35. The method of claim 33, wherein said estradiol is notderivatized prior to mass spectrometry.
 36. The method of claim 33,wherein said liquid chromatography is high performance liquidchromatography (HPLC).
 37. The method of claim 33, wherein saidpurifying further comprises high turbulence liquid chromatography (HTLC)followed by high performance liquid chromatography (HPLC).
 38. Themethod of claim 33, wherein said acidifying agent is formic acid. 39.The method of claim 33, wherein said estradiol ions comprise one or moreions selected from the group consisting of ions with a mass/charge ratioof 255.07±0.5, 159.20±0.5, and 133.20±0.5.
 40. The method of claim 33,wherein said ionizing comprises generating a precursor ion with amass/charge ratio of 255.07±0.5, and generating one or more fragmentions selected from the group consisting of ions with a mass/charge ratioof 159.20±0.5, and 133.20±0.5.
 41. The method of claim 33, wherein bothglucuronidated and non-glucuronidated estradiol present in said testsample are detected and measured by the method.
 42. The method of claim33, wherein said test sample is body fluid.
 43. A method for determiningthe amount of estradiol in a test sample said method comprising: (a)adding an agent to said test sample in an amount sufficient to freeestradiol from a protein that may be present in said test sample; (b)purifying estradiol from said test sample by liquid chromatography; (c)ionizing the purified estradiol from said test sample to produce one ormore ions detectable by tandem mass spectrometry; and (d) detecting theamount of the estradiol ion(s) by tandem mass spectrometry in negativeion mode, wherein the amount of the estradiol ion(s) is related to theamount of estradiol in said test sample.
 44. The method of claim 43,wherein said method has a limit of quantitation less than or equal to 80pg/mL.
 45. The method of claim 43, wherein said estradiol is notderivatized prior to mass spectrometry.
 46. The method of claim 43,wherein said liquid chromatography is high performance liquidchromatography (HPLC).
 47. The method of claim 43, wherein saidpurifying further comprises high turbulence liquid chromatography (HTLC)followed by high performance liquid chromatography (HPLC).
 48. Themethod of claim 43, wherein said agent is a salt.
 49. The method ofclaim 48, wherein said salt is ammonium sulfate.
 50. The method of claim43, wherein said estradiol ions comprise one or more ions selected fromthe group consisting of ions with a mass/charge ratio of 271.14±0.5,183.10±0.5, and 145.10±0.5.
 51. The method of claim 43, wherein saidionizing comprises generating a precursor ion with a mass/charge ratioof 271.14±0.5, and generating one or more fragment ions selected fromthe group consisting of ions with a mass/charge ratio of 183.10±0.5, and145.10±0.5.
 52. The method of claim 43, wherein both glucuronidated andnon-glucuronidated estradiol present in said test sample are detectedand measured by the method.
 53. The method of claim 43, wherein saidtest sample is body fluid.